Inflammation is a multifaceted process required to heal injuries and fight infections. However, chronic inflammation underlies various debilitating diseases including rheumatoid arthritis, asthma and heart disease. Glucocorticoids (GCs), which activate the glucocorticoid receptor (GR), are the most powerful anti- inflammatory drugs available and are unsurpassed in their ability to counter both acute and chronic inflammation. Despite their efficacy, continued GC use causes debilitating side effects including weight gain, diabetes, and Cushing's disease. In fact, rheumatoid arthritis patients are generally pulled off GC treatment after 3-6 months to avoid side effect development, despite GCs ability to slow disease progression dramatically in early stages. These side effects are the result of stimulating the transcriptional activity of GR, which controls the expression of both metabolic and inflammatory genes via distinct mechanisms. To activate transcription, GR directly binds to glucocorticoid response elements (GREs) to drive the transcription of metabolic and stress response genes. Conversely, GRs ability to repress genes is less well understood. Current theories include DNA-dependent and DNA-independent mechanisms. The first mechanism involves direct binding of GR to the newly discovered negative glucocorticoid response elements (nGREs). The DNA-independent mechanism involves GRs interaction with another well-characterized transcription factor, activator protein-1 (AP-1), which up-regulates the expression of inflammatory genes. To repress AP-1 activity, GR is proposed to bind to AP-1 through protein-protein interactions only and not through a direct DNA interaction. This mechanism, known as tethering, is the prevailing model for GR-mediated repression at inflammatory genes. However, recent discoveries regarding DNA-dependent GR-mediated transrepression reveal that the mutations used to develop the tethering hypothesis are inadequate. We show that these mutations prevent GR binding to nGREs and do not formally rule out direct DNA interaction as a major mechanism behind GR's ability to counter AP-1 signaling. Therefore, we hypothesize that GR-mediated repression of inflammatory genes occurs through an alternative mechanism involving direct DNA binding similar to nGREs. In Specific Aim 1, we will test for direct DNA interaction by GR at AP-1 binding sites within inflammatory gene promoters using a combination of biochemical techniques to determine the sequence specificity and the structural specificity of this interaction. In Specific Aim 2, we will test our proposed mechanism by which GR can repress inflammatory genes independent of AP-1 tethered interactions in cells. Resolving the mechanistic basis for direct GR-meditated transrepression at AP-1 sites will be critical to improve our understanding of GR's role in inflammatory diseases and could lay the foundation for new therapeutic development. Ideally, the best anti- inflammatory therapy would separate the metabolic effects, attributed to the side effects of GC use, from the desired anti-inflammatory effects. Our long-term goal is to determine if this type of glucocorticoid is possible.